Figure 4

Effects of cetuximab (100 μg ml⁻¹) alone or combined with trastuzumab (10 μg ml⁻¹), an anti-HER2 MAb, or PD153035 (0.1 μ M), an EGFR tyrosine kinase inhibitor, or PD98059 (25 μ M), a MAPK pathway inhibitor, on Caski and C33A cells. (A) Effects of cetuximab alone or combined to trastuzumab on the survival in CA. (B) Representative pictures of Caski and C33A cells under cetuximab and trastuzumab treatments in CA. (C and D) Western blotting analysis of the inhibition of EGF-induced phosphorylation of AKT and p44/42 (ERK1/2) by cetuximab alone or combined with trastuzumab in Caski and C33A cells, respectively. (E) Effects of cetuximab alone or combined with PD153035 on cell survival in CA. (F) Effects of cetuximab alone or combined with PD98059 on proliferation by MTT assays. (G) Western blotting analysis of the inhibition of EGF-induced phosphorylation of EGFR (Tyr1068), HER-2/neu, AKT and p44/42 (ERK1/2) by cetuximab alone or combined with PD153035. To visualise EGFR in C33A cells, 80 μg of protein were loaded on the SDS–PAGE gels as seen by the higher amount of the endogenous control, HSC-70. (H) Western blotting analysis of the inhibition of phosphorylation of p44/42 (Erk1/2) by cetuximab and PD98059. Student's t-test ^*P<0.05, when compared to control cells. (I) Proposed model of the effects of the combination of cetuximab with chemoradiation or trastuzumab or MAPK pathway inhibitor on CC cell lines. Ligand binding activates signalling through EGFR and triggers the AKT and MAPK pathways. The binding of cetuximab sensitises CC cells to chemoradiation (1) and to trastuzumab (2), leading to cell death independently of EGFR expression levels, but more dependent on EGFR-HER2 signalling. For cells in which activation of the MAPK pathway occurs also through EGFR independent mechanisms, cetuximab inhibition of EGFR sensitises them to PD98059 (3), a MAPK pathway inhibitor, leading to additive effects on the inhibition of cell proliferation.